The ultimate goal of the Mackall laboratory is to develop immune based therapies that induce potent and effective anti-tumor immune responses. Because chemotherapy is effective at reducing tumor bulk in nearly all pediatric malignancies, we are particularly focused upon the development of immune therapies that can be used in concert with chemotherapy to eradicate minimal residual disease. Accomplishing this goal requires not only a full understanding of immune reconstitution, but also a clear understanding of the biology of the host:tumor interface since effective antitumor immunotherapies must target appropriate antigens, expand the most effective cells and overcome negative regulatory influences which the tumor induces. The specific biology of the host:tumor interface is likely to vary from tumor to tumor, therefore the development of immunotherapy for specific diseases must entail study of the interaction of specific tumors with the immune system. As little is known regarding the immunobiology of pediatric sarcomas, our lab is currently actively engaged in studies which seek to advance our understanding of the host:tumor interface in general and the interface which exists in patients with pediatric sarcomas, in particular.Our previous work using lymphocytes harvested at the time of clinical presentation with Ewing's sarcoma showed that tumor-lytic T cells circulate in patients with Ewing's sarcoma, that these cells can be expanded and activated using anti-CD3 anti-4-1BB and that adoptive transfer of these cells to animals bearing Ewing's sarcoma xenografts can induce antitutmor effects in vivo. (Zhang et al., 2003). Based upon this, we have engaged in further studies examining the utility of 4-1BB mediated co-stimulation in expanding tumor lytic T cells. Through a collaboration with Carl June, Univ. of Pennsylvania, we have acquired artificial APCs which constitutively express 4-1BBL and have demonstrated that anti-CD3/4-1BBL ligand mediated expansion of CD8+ populations more effectively expands the memory T cell population for viral and flu antigens. Ultimately we hope to undertake adoptive transfer of cells which are expanded using 4-1BBL mediated costimulation in the context of immunotherapy trials. Toward this end, we have received funding through a Bench-to-Bedside grant to generate a master cell bank of artificial APCs. This represents the first step toward the development of adoptive immunotherapy for this disease and could potentially be adapted for use in other diseases as well.We have also continued studies of the biology of programmed cell death in Ewings sarcoma in an effort to identify potential blocks in death pathways which may modulate susceptibility to immune based therapies and/or to identify apoptosis inducing therapies which may have additive or synergistic effects on immunotherapy. These studies identified that agonists of TR2 (DR5) are sufficient to induce apoptosis in the vast majority of Ewing's sarcoma cells lines. In vivo, anti-TR2 moAbs and Apo2L (the natural trimeric ligand) both demonstrate significant activity as measured by shrinkage of primary tumors in xenografted mice. However, the potency of the response was modest in vivo when compared to the exquisite sensitivity observed in vitro. Using studies to determine whether TRAIL resistance was induced during the course of tumor growth in vivo, we observde TRAIL resistance of tumor explants during the first several passages ex vivo of tumor derived from either TRAIL agonist treated or non-treated animals. The TRAIL resistance was characterized by downregulation of TR2 and resistance was reversed using IFN gamma in vitro. When TRAIL receptor agonists were combined with IFN gamma in vivo, we observed an improved capacity for TRAIL based therapy to prevent metastatic disease.